1. Technical Field
The present invention is related to a method and a kit for detecting human exposure to genotoxic agents. More particularly, the present invention is related to immunoassays of antibodies to DNA adducted to a carcinogen or a mutagen, the presence of said antibodies in human serum being an indicator of human exposure to environmental carcinogens or mutagens and a kit containing various components for performing the assays.
2. State of the Art
Advanced technology and modern scientific development have produced several environmental pollutants, some being quite hazardous to health. Yet there are no objective means of monitoring human exposure to certain carcinogens or mutagens generally found in the industrial surroundings and in the atmosphere. The present invention provides an assay system and a kit for detecting the exposure of individuals to carcinogenic or mutagenic agents.
Of several hazardous elements prevailing in the atmosphere, benzo[a]pyrene (BP) is a ubiquitous chemical carcinogen found, for instance, in tobacco smoke, atmospheric pollution due to burning of fossil fuels, and a variety of foods (IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, 1983, World Health Organization, Lyon, Vol. 32, 211-224). In fact BP is so ubiquitous that it can be used as a reliable indicator of general exposure to other carcinogenic polycyclic aromatic hydrocarbons.
BP is a procarcinogen that requires metabolic activation, which results in its putative ultimate carcinogenic metabolite, 7.beta.,8.alpha.-dihydroxy-9.alpha.,10.alpha.)-epoxy-7,8, 9,10-tetrahydrobenzo[a]pyrene (BPDE) (Gelboin, Physiol. Rev. 60:1107-1166, 1980). The predominant DNA adducts formed from these compounds have been studied in experimental animals and in cultured human tissues and cells (Harris, et al., J. Cell. Biochem. 18:285-294, 1982; Jeffrey, et al., Nature, 269:348-350, 1977) and are found to be highly variable probably due to differences in metabolic enzymes.
DNA adduct levels are also dependent on DNA repair rates. Although rates for excision DNA repair vary several fold among people (Setlow, Human Carconogenesis, pp. 231-254, 1983), the interindividual variation in the DNA repair rates of these BPDE-DNA adducts in humans is not known. Therefore, the amount of adducts measured at any timepoint is dependent on many factors, including exposure to BP, its absorption and transport, the metabolic balance between activation and deactivation of BP and on the capacity of the cells to repair DNA adducts.
It should be noted that the methods of measuring DNA damage per se are known but heretofore it was not suspected that there might be antibodies present in human sera induced by DNA-carcinogen adducts and DNA damaged by oxidative stress caused by carcinogens (Cerutti. Science 227:375-281, 1985). Therefore, detection of antibodies in human sera to carcinogen-induced DNA damage, e.g., carcinogen-DNA adducts by immunoassays, was never thought of. As disclosed herein for the first time, the demonstration of the presence of antibodies to DNA-carcinogen adducts in human sera, provides an objective and reliable means to monitor human exposure to patho- biologically effective doses of chemical and physical carcinogens. This internal dosimeter of genetic damage reflected in terms of induced antibodies has unique advantages over merely measuring DNA damage directly in human cells.
First, the determination of just the DNA damage may reflect only the residual DNA damage, i.e., the DNA remaining after repair. Hence, such measurement may indicate only the present status of DNA damage, i.e., hours to days and this may only be an indicator of recent exposure to carcinogens. In contrast, the immunological memory found in lymphocytes, and the antibodies found in human sera could detect human exposure to carcinogens far into the past, i.e., years to decades. The finding of such antibodies in human sera was indeed unexpected because the intracellular DNA should normally remain protected from the immune systems, hence not normally expected to be available to form an antigenic entity.